Exploring liquid-liquid phase separation in vitro and in vivo using multimodal nonlinear optical imaging.

Area of Science:

• Biophysics
• Cell Biology
• Molecular Imaging

Background:

• Liquid-liquid phase separation (LLPS) forms biomolecular condensates like P granules.
• P granules are crucial for germline development and gene regulation in C. elegans.
• Label-free imaging offers a non-invasive method for visualizing cellular structures.

Purpose of the Study:

• To demonstrate label-free visualization of liquid droplets (LqDs) using multimodal nonlinear optical imaging.
• To quantify RNA concentration within LqDs in vitro and in vivo.
• To assess the feasibility of detecting P granules in C. elegans without exogenous labels.

Main Methods:

• Multimodal nonlinear optical imaging techniques were employed for label-free visualization.
• In vitro experiments utilized polymerized adenine [poly(A)] to establish detection limits.
• In vivo imaging was performed on wild-type and GFP-tagged Caenorhabditis elegans (C. elegans).

Main Results:

• In vitro, significantly higher poly(A) concentrations were detected in LqDs, with a limit of detection (LoD) of 32 mg/mL.
• In vivo imaging of C. elegans P granules did not yield clear RNA-specific signals.
• Observed RNA concentrations in P granules were below the established in vitro LoD.

Conclusions:

• Label-free nonlinear optical imaging can visualize LqDs in vitro and quantify specific molecules like poly(A).
• The RNA concentration within C. elegans P granules is lower than anticipated, posing challenges for label-free detection.
• Further advancements in imaging sensitivity are needed for robust in vivo label-free RNA detection in P granules.